五种出血热病毒重组酶聚合酶等温扩增方法的建立

发布时间：2018-05-26 04:46

本文选题：重组酶聚合酶检测 + 等温扩增　；参考：《解放军医学杂志》2017年06期

【摘要】：目的建立五种出血热相关病毒,即扎伊尔型埃博拉病毒、苏丹型埃博拉病毒、马尔堡病毒、拉沙病毒和黄热病毒的一步法重组酶聚合酶等温扩增(RPA)技术,为病原体的确定提供现场快速检测方法。方法通过基因组序列分析,选择每种病毒的特异核酸片段作为检测靶基因,针对靶基因序列设计RPA检测的引物和探针;取系列稀释的模板基因进行RPA检测,确定其灵敏性;取出血热相关病毒核酸为模板进行RPA检测,确定其特异性;验证不同温度反应条件(37~42℃)对检测结果的影响。结果五种出血热病毒的RPA反应体系均可有效扩增靶基因,灵敏性为1.5×102~1.5×103copies/反应,与其他出血热相关病毒的核酸无交叉反应;RPA实验在37~42℃均可实现靶基因的有效扩增。结论建立了五种出血热病毒的RPA等温扩增体系,该体系反应快速,温度范围宽,适用于现场快速检测。
[Abstract]:Objective to establish a one step recombinant enzyme polymerase isothermal amplification (RPA) technique for five hemorrhagic fever associated viruses, namely, Zairian Ebola virus, Sudan Ebola virus, Marburg virus, Lassa virus and yellow fever virus. To provide a rapid field detection method for the determination of pathogens. Methods by genomic sequence analysis, specific nucleic acid fragments of each virus were selected as target genes, primers and probes for RPA detection were designed for target gene sequences, and RPA was used to detect a series of diluted template genes to determine their sensitivity. The nucleic acid of haemorrhagic fever associated virus was used as a template for RPA detection to determine its specificity, and to verify the effect of different temperature reaction conditions on the detection results. Results the five RPA reaction systems of hemorrhagic fever virus could amplify the target gene effectively, the sensitivity was 1.5 脳 10 ~ 2 ~ (-1) 脳 103copies/, and the target gene could be amplified effectively without cross-reaction with the nucleic acid of other hemorrhagic fever associated virus. Conclusion five RPA isothermal amplification systems of haemorrhagic fever virus have been established. The system is suitable for rapid detection of hemorrhagic fever virus in the field because of its rapid reaction and wide temperature range.
【作者单位】：安徽医科大学研究生学院;军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室;
【基金】：国家自然科学基金(81501789) 军队重大专项课题(AWS15J006) 国家重点实验室课题(SKLPBS1415)~~
【分类号】：R440;R512.8

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